γh2ax

The Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktail (Beyotime, Shanghai, China) was used to extract cellular protein. The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China) was use to extract subcellular protein. The extracted samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in order to separate the component proteins before they were immunoblotted onto PVDF membranes. 5% nonfat dry milk in TBST was used to block membranes. The appropriate primary antibodies were then added to the membranes and allowed to incubate. This was followed by incubation with HRP-conjugated secondary antibodies. Western blotting analysis was performed using anti-RACGAP1, LIG3, γH2A.X, p-ATM, p-ATR, p-CHEK1 and p-CHEK2, PARP1, caspase3 and c-caspase3, PAR, GAPDH and Histone H3 antibodies (p-ATM, p-ATR, p-CHEK1, p-CHEK2 and PARP1 were supplied by ABclonal, MA, USA, others were from Abcam, MA, USA).

CRC samples and cells were lysed in RIPA buffer supplemented by protease and phosphatase inhibitors (TargetMol, America) and incubated on ice for 30 min. The supernatant was collected after discarding the sedimentation. The denatured proteins were added to the chamber for electrophoresis in SDS-PAGE and subsequently transferred onto PVDF membranes by electrotransfer. After blocking with 3% BSA for 1 h, the membranes were incubated with diluted primary antibody overnight at 4 °C. On the following day, after discarding the primary antibody, the samples were washed three times with TBST and incubated with diluted secondary antibody for 1 h at room temperature. The immune complexes were detected via an enhanced chemiluminescence system (Life Tec, America). Analysis and quantification of the bands were performed by ImageJ software (Version 11). The primary antibodies involved in this work include KIF11 (1:1000, CST, America), γH2AX (1:1000, Abclonal, China), caspase 3/cleaved caspase 3 (1:1000, CST, America), caspase 9/cleaved caspase 9 (1:1000, CST, America), Bax (1:1000, Abclonal, China), Bad (1:1000, Abclonal, China), and GAPDH (1:1000, Abclonal, China). Secondary antibodies were purchased from Abclonal, China.

Primary antibodies against NDUFS1 (no. 12444), NDUFA9 (no. 20312-1-AP), NDUFV1 (no. 11238), NDUFV2 (no. 5301), KU80 (no. 6389), KU70 (no. 10723), RAD51 (no. 14961), PARP-1 (no. 13371), PDP1 (no. 21176),PDK1 (no. 10026), β-actin (no. 60008), and Histone3 (no.17168) were obtained from Proteintech and γ-H2AX (no. AP0099), PDHA1 (no. A17432) were obtained from Abclonal and p-PDH (no.ab115343), HIF1a (no. ab179483) were obtained from Abcam, and Acetyl-Histone H3 (Lys9) (no. 9649 s), Acetyl-Histone H3 (Lys56) (no. 4243 s), were obtained from CST in this study.

Below mentioned is the list of chemicals and antibodies used in this study: Anti-Klotho (A12028) was purchased from Abclonal (Woburn, MA, USA), anti-p21 (sc-6246) and anti-TERT (sc-377511) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and γ -H2AX from Abclonal. The secondary antibodies (HRP-conjugated anti-mouse and anti-rabbit) were purchased from Invitrogen. Doxorubicin hydrochloride was purchased from Sigma-Aldrich (44583).